Difference between revisions of "Part:BBa K3416007"
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=Introduction= | =Introduction= | ||
[[File:T--Vilnius-Lithuania--FFlogo.png|80px|right|FlavoFlow]] | [[File:T--Vilnius-Lithuania--FFlogo.png|80px|right|FlavoFlow]] | ||
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Vilnius-Lithuania iGEM 2020 project [https://2020.igem.org/Team:Vilnius-Lithuania <b>FlavoFlow]</b>includes three goals towards looking for <i>Flavobacterium</i> disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. | Vilnius-Lithuania iGEM 2020 project [https://2020.igem.org/Team:Vilnius-Lithuania <b>FlavoFlow]</b>includes three goals towards looking for <i>Flavobacterium</i> disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. | ||
This part was used for the third goal- prevention - of the project FlavoFlow. | This part was used for the third goal- prevention - of the project FlavoFlow. | ||
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| − | + | =Biology= | |
| − | <b>Gliding motility protein</b> (GldJ) is a protein that is required for gliding motility. Gliding motility is a process of bacterial movement on surfaces used by several bacterial species. Instead of involving flagellae or pili, gliding motility is applied by complex machinery< | + | <b>Gliding motility protein</b> (GldJ) is a protein that is required for gliding motility. Gliding motility is a process of bacterial movement on surfaces used by several bacterial species. Instead of involving flagellae or pili, gliding motility is applied by complex machinery<ref>McBride, M. J. Bacterial Gliding Motility: Multiple Mechanisms for Cell Movement over Surfaces. <i>Annu. Rev. Microbiol.</i> <b>55</b>, 49–75 (2001).</ref>. This mechanism is based on motors associated with the cell surface, the inner membrane and mechanical force<ref>Nan, B. Bacterial Gliding Motility: Rolling Out a Consensus Model. <i>Current Biology</i> <b>27</b>, R154–R156 (2017).</ref>. Gliding motility machinery is composed of Gld and Spr proteins. There is known that Spr are cell-surface adhesins, while an exact function of Gld protein remains unclear. It is estimated that it plays an essential role in gliding processes. Gld could be associated with the SprB filaments, but GldJ label separate non-filamentous structures in the cell fraction<ref>Nakane, D., Sato, K., Wada, H., McBride, M. J. & Nakayama, K. Helical flow of surface protein required for bacterial gliding motility. <i>Proceedings of the National Academy of Sciences</i> <b>110</b>, 11145–11150 (2013).</ref>. |
Gliding motility is common in bacteroidetes, of which <i>Flavobacterium</i> is a member of. Cells from some <i>Flavobacterium</i> species can glide over surfaces using this mechanism. | Gliding motility is common in bacteroidetes, of which <i>Flavobacterium</i> is a member of. Cells from some <i>Flavobacterium</i> species can glide over surfaces using this mechanism. | ||
| − | Most of the research on GldJ was done using <i>F. johnsoniae</i>, its mutants without <i>gldJ</i> gene formed non-spreading colonies and individual cells were immobile< | + | Most of the research on GldJ was done using <i>F. johnsoniae</i>, its mutants without <i>gldJ</i> gene formed non-spreading colonies and individual cells were immobile<ref>McBride, M. J. & Braun, T. F. GldI Is a Lipoprotein That Is Required for <i>Flavobacterium johnsoniae</i> Gliding Motility and Chitin Utilization. <i>JB</i> <b>186</b>, 2295–2302 (2004).</ref>. By using gliding motility, cells can move up to 10 μm/s. This movement with GldJ involvement supports a model of gliding motility in which motors anchored to the cell wall propel cell surface adhesins<ref>Nelson, S. S., Bollampalli, S. & McBride, M. J. SprB Is a Cell Surface Component of the <i>Flavobacterium johnsoniae</i> Gliding Motility Machinery. <i>JB</i> <b>190</b>, 2851–2857 (2008).</ref>. |
| − | GldJ is a unique immunogenic protein from the family of bacterial outer membrane proteins (OMPs) and it has already been used for vaccine development. OMPs receive much attention as vaccine candidates because they have well-exposed epitopes on the cell surface that allow them to be recognized easily by the host immune system< | + | GldJ is a unique immunogenic protein from the family of bacterial outer membrane proteins (OMPs) and it has already been used for vaccine development. OMPs receive much attention as vaccine candidates because they have well-exposed epitopes on the cell surface that allow them to be recognized easily by the host immune system<ref>Ningqiu, L. et al. An outer membrane protein, OmpK, is an effective vaccine candidate for <i>Vibrio harveyi</i> in Orange-spotted grouper (Epinephelus coioides). <i>Fish & Shellfish Immunology</i> <b>25</b>, 829–833 (2008).</ref>. |
| − | Original research of GldJ immunogenic properties was done by amplifying the coding sequence using primers to amplify directly from F. columnare. Primers include appropriate enzymatic restriction sites at the 5′ ends for cloning. PCR product was cloned into plasmid pET-28a(+) and expressed in <i>E. coli</i> BL21 (DE3) strain. The recombinant protein was purified using the Ni-NTA based affinity chromatography. After vaccination with GldJ protein, cumulative mortality of the immunized fish with GldJ was 23.3%, compared with control - 83.3%. It shows great potential because the relative percent survival of the groups immunized with recombinant GldJ was 72% when compared to control fish. Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of <i>F. columnare</i>< | + | Original research of GldJ immunogenic properties was done by amplifying the coding sequence using primers to amplify directly from F. columnare. Primers include appropriate enzymatic restriction sites at the 5′ ends for cloning. PCR product was cloned into plasmid pET-28a(+) and expressed in <i>E. coli</i> BL21 (DE3) strain. The recombinant protein was purified using the Ni-NTA based affinity chromatography. After vaccination with GldJ protein, cumulative mortality of the immunized fish with GldJ was 23.3%, compared with control - 83.3%. It shows great potential because the relative percent survival of the groups immunized with recombinant GldJ was 72% when compared to control fish. Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of <i>F. columnare</i><ref>Luo, Z. et al. Immunogenic proteins and their vaccine development potential evaluation in outer membrane proteins (OMPs) of Flavobacterium columnare. <i>Aquaculture and Fisheries</i> <b>1</b>, 1–8 (2016). |
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===References=== | ===References=== | ||
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Latest revision as of 22:16, 16 December 2020
Introduction
Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This part was used for the third goal- prevention - of the project FlavoFlow.
Contents
Biology
Gliding motility protein (GldJ) is a protein that is required for gliding motility. Gliding motility is a process of bacterial movement on surfaces used by several bacterial species. Instead of involving flagellae or pili, gliding motility is applied by complex machinery[1]. This mechanism is based on motors associated with the cell surface, the inner membrane and mechanical force[2]. Gliding motility machinery is composed of Gld and Spr proteins. There is known that Spr are cell-surface adhesins, while an exact function of Gld protein remains unclear. It is estimated that it plays an essential role in gliding processes. Gld could be associated with the SprB filaments, but GldJ label separate non-filamentous structures in the cell fraction[3]. Gliding motility is common in bacteroidetes, of which Flavobacterium is a member of. Cells from some Flavobacterium species can glide over surfaces using this mechanism. Most of the research on GldJ was done using F. johnsoniae, its mutants without gldJ gene formed non-spreading colonies and individual cells were immobile[4]. By using gliding motility, cells can move up to 10 μm/s. This movement with GldJ involvement supports a model of gliding motility in which motors anchored to the cell wall propel cell surface adhesins[5]. GldJ is a unique immunogenic protein from the family of bacterial outer membrane proteins (OMPs) and it has already been used for vaccine development. OMPs receive much attention as vaccine candidates because they have well-exposed epitopes on the cell surface that allow them to be recognized easily by the host immune system[6]. Original research of GldJ immunogenic properties was done by amplifying the coding sequence using primers to amplify directly from F. columnare. Primers include appropriate enzymatic restriction sites at the 5′ ends for cloning. PCR product was cloned into plasmid pET-28a(+) and expressed in E. coli BL21 (DE3) strain. The recombinant protein was purified using the Ni-NTA based affinity chromatography. After vaccination with GldJ protein, cumulative mortality of the immunized fish with GldJ was 23.3%, compared with control - 83.3%. It shows great potential because the relative percent survival of the groups immunized with recombinant GldJ was 72% when compared to control fish. Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F. columnare[7].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 709
Illegal XhoI site found at 153 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ McBride, M. J. Bacterial Gliding Motility: Multiple Mechanisms for Cell Movement over Surfaces. Annu. Rev. Microbiol. 55, 49–75 (2001).
- ↑ Nan, B. Bacterial Gliding Motility: Rolling Out a Consensus Model. Current Biology 27, R154–R156 (2017).
- ↑ Nakane, D., Sato, K., Wada, H., McBride, M. J. & Nakayama, K. Helical flow of surface protein required for bacterial gliding motility. Proceedings of the National Academy of Sciences 110, 11145–11150 (2013).
- ↑ McBride, M. J. & Braun, T. F. GldI Is a Lipoprotein That Is Required for Flavobacterium johnsoniae Gliding Motility and Chitin Utilization. JB 186, 2295–2302 (2004).
- ↑ Nelson, S. S., Bollampalli, S. & McBride, M. J. SprB Is a Cell Surface Component of the Flavobacterium johnsoniae Gliding Motility Machinery. JB 190, 2851–2857 (2008).
- ↑ Ningqiu, L. et al. An outer membrane protein, OmpK, is an effective vaccine candidate for Vibrio harveyi in Orange-spotted grouper (Epinephelus coioides). Fish & Shellfish Immunology 25, 829–833 (2008).
- ↑ Luo, Z. et al. Immunogenic proteins and their vaccine development potential evaluation in outer membrane proteins (OMPs) of Flavobacterium columnare. Aquaculture and Fisheries 1, 1–8 (2016).